Acetyl CoA carboxylase (ACC), fatty acid synthetase (FAS), pyruvate carboxylase (PC) and malic enzyme (ME) play important functions in the biosynthesis of long chain saturated fatty acids of 3T3-L adipocytes. Both ACC and PC contain covalently linked biotin which functions as a carboxyl carrier during the reactions catalyzed by these carboxylases. PC is located in mitochondria whereas ACC, FAS and ME are found in the cytosol. When confluent 3T3-L cells are maintained on a biotin-deficient medium they still differentiate to adiposytes. However, as a result of this nutritional stress they accumulate apo-PC. In addition, the activities of ME and glycerophosphate acyltransferase decrease and ACC is repressed. Because of the morphologic and biochemical similarilty to mature mammalian adipocytes, 3T3-L adipocytes provide a suitable model system to investigate various aspects of lipogenesis and its control under a variety of rigidly controlled conditions. The proposed studies will focus on the following points: a) Does biotin deficiency cause repression of ACC and perhaps of other lipogenic enzymes found in the cytosol? b) How does biotin regulate the activity of ME? c) Where does apo-PC accumulate (mitochondria or cytosol) under biotin-deficient conditions? d) Does there exist a precursor of PC? e) Which form of PC (apo-, holo-, pre-apo, or pre-holo) is transported and packaged in mitochondria of 3T3-L adipocytes?